The regulation of cytokine gene expression in Th2 cells involves both Th2 cell-specific and non-Th2 cell-specific transcription factors. While much is known about the roles of each of the transcription factors, how their actions are coordinated and integrated is poorly understood, and the biochemical/enzymatic processes initiated by these factors are largely undefined. We propose to use the newly developed tandem affinity purification (TAP) technology to isolate protein complexes associated with these transcription factors in Th2 cells. The proteins in the complexes will be identified by matrix assisted laser desorpsion/ionization-time of flight mass spectrometric (MALDI-TOF MS) protein finger printing analysis. Based on their association with the same complex we will determine the relationships among the transcription regulatory molecules. From compositions of the complexes, we also wish to predict their biochemical functions and relationships. The information gained will allow us to formulate hypotheses for an integrated network of cytokine gene regulation, which will provide a roadmap for future experimental studies. The TAP technology offers purification of protein complexes in their native forms to near homogeneity, whereas MALDI-TOF MS identifies protein in picomolar quantity without prior knowledge of the proteins. In combination, they provide great advantages over conventional technologies for protein analysis. Th2 cell-specific new factors in the complexes will be introduced into Th1 cells with GATA-3 to test whether they can cooperate with GATA-3 to induce optimal expression of Th2 cytokines and repress IFN-? expression. In addition, dominant-negative mutants of these factors will be expressed in uncommitted CD4 T cells to test their abilities to attenuate GATA-3-mediated Th2 differentiation. The studies in this proposal are extension of the study in our parent grant to isolate molecules associated with GATA-3 to understand the mechanisms of cytokine gene regulation in Th2 cells.